Alternate polypurine tracts affect rous sarcoma virus integration in vivo

Abstract

When the endogenous polypurine tract (PPT) of the Rous sarcoma virus (RSV)-derived vector RSVP(A)Z was replaced with alternate retroviral PPTs, the fraction of unintegrated viral DNA with the normal consensus ends significantly decreased and the retention of part of the PPT significantly increased. If the terminus of the U3 long terminal repeat (LTR) is aberrant, RSV integrase can correctly process and integrate the normal U5 LTR into the host genome. However, the canonical CA is not involved in joining the aberrant U3 LTR to the host DNA, generating either large duplications or deletions of the host sequences instead of the normal 5- or 6-bp duplication.

FIG. 1.

Schematic drawing showing the structures of the RSVP vectors. (A) Sequences of the alternate PPTs. (B) Schematic representation of the circle junctions. U3 is indicated by white boxes with vertical black bars; R is indicated by black boxes; U5 is shown by white boxes with horizontal black bars; PPT insertions are indicated by a white box. The terminal sequence of U3 is shown; the PPT sequence is represented by Xs. The sites of the RNase H cleavages are indicated by black arrows. (C) 2-LTR circle junctions isolated from infected cells. (D) Schematic numbering of the nucleotide positions at the ends of the proviruses. The last nucleotide on each end of a normal provirus is number 1.

FIG. 2.

Provirus flanked by an inversion of host sequences. (A) Structure of a provirus. U3 is indicated by white boxes with vertical black bars; R is indicated by black boxes; U5 is shown by white boxes with horizontal black bars. Six nucleotides at the viral/host DNA junctions are shown. The orientations of the host sequences are indicated by black arrows. (B) Normal integration event. A 5- to 6-bp duplication is indicated by white boxes with vertical black bars. (C) Model for the generation of a provirus flanked by an inversion of host sequences. The deletion in the 5′ LTR is indicated by a jagged black end. A host DNA is indicated by thick black lines, and a viral DNA is indicated by thin black lines. The joinings between viral DNA and host DNA are indicated by black circles.

FIG. 3.

Structure of direct duplications of U3 sequences in the U3 LTR. U3 is indicated by white boxes with vertical black bars; R is indicated by black boxes; U5 is shown by white boxes with horizontal black bars. Direct duplications are indicated by white arrows, and the sizes of the duplications are shown. The detailed sequences at the junctions are shown in white boxes, and microhomologies are indicated by bold letters. Normal target duplications are indicated by black arrows.